Human and animal-infective trypanosomes have major medical and veterinary consequences for equatorial Africa and Latin America. Failure of the immune system to eliminate infections with the Salivarian trypanosomes is largely attributable to the phenomenon of antigenic variation. Each trypanosome is covered by a surface coat consisting of a closely packed monolayer of about 10 million molecules of a single member of a large family of variant surface glycoproteins (VSGs). VSGs are anchored to the trypanosome surface membrane via a covalently attached glycosylphosphatidylinositol (GPI) moiety. The integrity of the VSG coat is essential for trypanosome survival. Trypanosoma brucei also contains a GPI-specific phospholipase-C (GPIPLC). This situation suggests that VSGs are GPI anchored for very specific but so far elusive reasons, perhaps related to VSG turnover. Our previous studies led to the elucidation of the VSG-linked GPI structure, the structural characterization of GPI precursors, and an outline knowledge of the pathway of GPI biosynthesis. These studies are of general scientific interest since all eukaryotic cells have subsequently been found to express GPI-anchored cellsurface glycoproteins with diverse functions. The core structure of the GPI anchor appears to have been conserved during evolution. In trypanosomes, the major surface glycoproteins of bloodstream-form and procyclic T. brucei and of the infective stages of Leishmania and T. cruzi are GPI anchored. All of these glycoproteins probably have critical roles in infectivity and virulence. This continuation proposal will focus on the possible function of trypanosome GPI anchors through an exploration of the roles of the GPIPLC, the pathways that determine whether GPI anchors will be PIPLC-sensitive or resistant, the signals for GPI addition to proteins in trypanosomes, and GPI galactosylation. A primary objective of these studies is to identify aspects of GPI structure and function that may be distinct from host pathways. So far, GPIPLC has only been convincingly purified from T. brucei and its gene has been cloned. Its intracellular location is uncertain and its role in VSG metabolism is unknown. We propose genetic and organellar fractionation approaches to obtain more information on the role and subcellular location of GPIPLC in trypanosomes. Now that procyclic and bloodstream-form trypanosomes can routinely be transfected and cultured in vitro, we propose to use genetic approaches to further explore the carboxy-terminal amino acid sequence requirements for GPI anchoring of VSGs and PARP and determine whether the remarkably conserved VSG carboxy-terminal sequences indicate a very specific sequence requirement for GPI anchoring in trypanosomes. These studies may open up new in vitro approaches to identifying components of the GPI transfer machinery.